The invention relates to new modified neomycin-phosphotransferase genes and their use in selection methods for high-producing recombinant cells. Accordingly, the present invention also relates to new expression vectors which contain a modified neomycin-phosphotransferase gene, preferably combined with a gene of interest functionally linked to a heterologous promoter. The invention further relates to methods of preparing heterologous gene products using the corresponding high-producing recombinant cells.
Mammalian cells are the preferred host cells for the production of complex biopharmaceutical proteins as the modifications carried out post-translationally are compatible with humans both functionally and pharmacokinetically. The main relevant cell types are hybridoma, myeloma CHO (Chinese Hamster Ovary) cells and BHK (Baby Hamster Kidney) cells. The cultivation of the host cells is increasingly carried out under serum- and protein-free production conditions. The reasons for these are the concomitant cost reduction, the reduced interference in the purification of the recombinant protein and the reduction in the potential for the introduction of pathogens (e.g. prions and viruses). The use of CHO cells as host cells is becoming more widespread as these cells adapt to suspension growth in serum- and protein-free medium and are also regarded and accepted as safe production cells by the regulatory authorities.
In order to produce a stable mammalian cell line which expresses a heterologous gene of interest (GOI), the heterologous gene is generally inserted in the desired cell line together with a selectable marker gene such as e.g. neomycin phosphotransferase (NPT) by transfection. The heterologous gene and the selectable marker gene can be expressed in a host cell starting from one individual or separate co-transfected vectors. Two to three days after transfection the transfected cells are transferred into medium containing a selective agent, e.g. G418 when using neomycin phosphotransferase-gene (NPT gene), and cultivated for some weeks under these selective conditions. The emerging resistance cells which have integrated the exogenous DNA can be isolated and investigated for expression of the desired gene product (of the GOI).
A major problem in establishing cell lines with a high expression of the desired proteins arises from the random and undirected integration of the recombinant vector into transcriptionally-active or transcriptionally-inactive loci in the host cell genome. As a result a population of cells is obtained which have completely different expression rates of the heterologous gene, the productivity of the cells generally following normal distribution. In order to identify cell clones which have a very high expression of the heterologous gene of interest it is therefore necessary to examine and test a large number of clones, which is time consuming, labour intensive and expensive. Improvements to the vector system used for transfection therefore set out to increase the proportion of high producers in the transfected cell population by suitable selection strategies and thereby reduce the expenditure and work involved in clone identification. The development of such an expression system is the subject of the present invention.
The amino glycoside-3′-phosphotransferase II enzyme (neomycin-phosphotransferase) (EC27195) the gene of which is transposon 5-associated in Escherichia coli is used as a selectable marker in a number of organisms (e.g. bacteria, yeasts, plants and mammalian cells). This enzyme confers resistance to various aminoglycoside antibiotics such as neomycin, kanamycin and G418, by inactivating the antibiotics by transferring the terminal phosphate from ATP to the 3′ hydroxyl group of the aminohexose ring I. In addition to the wild-type neomycin phosphotransferase some mutants are known which have reduced phosphotransferase activity and hence reduced resistance to aminoglycoside antibiotics in bacteria (Blázques, J. et al., Molecular Microbiology 1991, 5(6), 1511-1518; Kocabiyik, S. et al., Biochem Biophys Res Commun 1992, 185(3), 925-931; Yenofsky, R. L. et al., Proc Natl Acad Sci USA 1990, 87, 3435-3439) and in slices of leaf from tobacco (Yenofsky, R. L. et al., Proc Nat/Acad Sci USA 1990, 87, 3435-3439).
One of these mutants (Glu182Asp) was used as a marker for selecting embryonic stem cells, the neomycin phosphotransferase gene being integrated into the c-myc gene by targeted homologous recombination (gene targeting) (Hanson, K. D. et al., Mol Cell Biol 1995, 15(1), 45-51). The authors restrict themselves to the use of the modified enzyme for gene targeting.
Patent application WO 99/53046 describes the expression of a modified neomycin phosphotransferase gene (Asp261Asn) in production-relevant mammalian cells. The authors describe a non-cloning method for expression of a gene of interest in mammalian cells. By cotransfection of the cells with three individual DNA fragments which code for a promoter element, a gene of interest and a selectable marker coupled with an IRES (“Internal ribosomal entry site”) element, it is possible to deliberately grow cells, under selection pressure, in which all three DNA fragments are combined as a functional bicistronic transcription unit (promoter gene of interest-IRES-neomycin-phosphotransferase gene). The arrangement of the elements only occurs in the transfected cell, so that only a few cells show the correct arrangement of the elements. Moreover, after gene amplification, using an amplifiable selectable marker, no high producing clones can be generated. After repeated selection and gene amplification the cells generated exhibited at most 6 pg of protein per cell per day (6 pg/cell/day).
None of the publications discloses modified neomycin phosphotransferase genes with particular suitability for the preparation of a high expression vector system for mammalian cells which makes it possible to develop high producing cells in order to prepare recombinant biopharmaceutical proteins which contain one or more complete functional transcription units both for one or more genes of interest and also for a modified neomycin phosphotransferase gene with reduced antibiotic resistance. The DNA construct described in WO 99/53046 contains only a promoter-less neomycin gene functionally linked to the gene for dihydrofolate reductase (DHFR).
There is therefore a need to make suitable modified neomycin phosphotransferase genes available, particularly for the development of corresponding high expression vector systems for biopharmaceutical processes. The problem of the present invention was therefore to provide corresponding new modified neomycin phosphotransferase genes, expression vectors which contain a modified neomycin phosphotransferase gene and a gene of interest functionally linked to a heterologous promoter, a method of selection for high producing recombinant cells, preferably for mammalian cells, and a process for producing heterologous gene products.
Surprisingly, within the scope of the present invention, it has been possible to produce and identify new modified highly selective neomycin phosphotransferase genes which are characterised by their particular suitability for the selection of high producing cells.